Despite advances in brain tumor therapy, little progress has been made to improve the prognosis of patients with glioblastoma. Invasive cells remaining after surgical resection significantly contribute to the demise of the patient without evidence of a discernable mass effect or recurrent bulk disease. Thus, successful treatment requires targeting the invasive portion of the tumor in addition to the core lesion, which is unmet by current treatment regimens. This project exploits the TWEAK-Fn14 targets and signaling pathways as an innovative approach to treat glioma invasion. We have developed cell-based assays and reagents for high throughput screening (HTS) of chemical libraries to identify drug-like inhibitors of TWEAK-Fn14 interactions. Our cell-based assay will interrogate both orthotopic and allosteric modulators that have a functional consequence throughout the TWEAK-Fn14 signaling pathway. Using the resources available through the Prebys Center at Sanford-Burnham, our objective is to perform a cell-based HTS format of the NIH's structurally diverse chemical library to identify drug-like compounds that inhibit the TWEAK-Fn14 signaling pathway. Selectivity profiles will be made using in-hand secondary cell-based TWEAK-Fn14-NF-?B driven luciferase assays and other TNF?-TNFR - driven NF-?B luciferase assays. Mechanism of action (MOA) studies will assess the impact of validated hits along the TWEAK-Fn14 signaling pathway. Finally, structure- activity relationship (SAR) by purchase of analog of validated hit series will be accomplished, along with supporting pharmacological characterization on optimized probe(s). One emphasis will be placed on identifying high affinity small molecules that possess appropriate physicochemical properties to promote passage across the blood-brain-barrier (BBB) and delivery into the brain parenchyma. The goal o the project is to identify research tool compounds targeting TWEAK-Fn14 for future hit-to-lead optimization campaigns directed towards development of drug leads, the following Specific Aims are proposed: Aim 1: Implement a cell-based reporter assay and perform a HTS of the NIH MLSMR >365,000 collection for modulators of the TWEAK-Fn14 signaling pathway; Aim 2: Confirm initial activity and validate hits for potency and selectivity/non-cytotoicity with existing secondary cellular assays for TWEAK-Fn14 pathway dependence and general cytotoxicity; Aim 3: Perform a limited structure-activity relationship (SAR) elucidation through SAR-by-purchase of available commercial analogs through the hit validation cascade of secondary assays to select the most potent and selective chemical probe; Aim 4: Perform authentic biological assays and tertiary assays on validated scaffolds to elucidate the potential mechanisms of action, blockade of cellular migration, invasion and survival and evaluation of efficacy in other cell types and clinically relevant primary cells~ Aim 5. Perform ADME/T and in vitro BBB profiling on the most promising tractable probe(s) and scale up (25 - 50 mg) for a limited rodent PK study and future proof of concept and research studies.